Rumen methanogens: a review

123

Indian J Microbiol (September 2010) 50:253–262 253

REVIEW

Rumen methanogens: a review

S. K. Sirohi · Neha Pandey · B. Singh · A. K. Puniya

Received: 16 July 2008 / Accepted: 6 August 2008

© Association of Microbiologists of India 2010

Indian J Microbiol (September 2010) 50:253–262

DOI: 10.1007/s12088-010-0061-6

Abstract The Methanogens are a diverse group of organ-

isms found in anaerobic environments such as anaerobic

sludge digester, wet wood of trees, sewage, rumen, black

mud, black sea sediments , etc which utilize carbon dioxide

and hydrogen and produce methane. They are nutritionally

fastidious anaerobes with the redox potential below –300

mV and usually grow at pH range of 6.0–8.0 [1]. Sub-

strates utilized for growth and methane production include

hydrogen, formate, methanol, methylamine, acetate, etc.

They metabolize only restricted range of substrates and

are poorly characterized with respect to other metabolic,

biochemical and molecular properties.

Keywords Rumen methanogens · Methanogens ·

Methane

Introduction

The domain Archaea consists of Crenarchaeota and Eu-

ryarchaeota. The Crenarchaeota are obligate thermophilic

organisms and mostly metabolize elemental sulphur. Sul-

folobus acidocaldarius is a representative species of this

domain which thrives in acid hot environments such as

hot springs. The Euryarchaeota contains methane form-

ing, extremely halophilic, sulfate reducing, and extremely

thermophilic sulfur metabolizing spp. Methanobacterium

formicicum which produces methane gas from hydrogen

gas and carbon dioxide, found in fresh water sediments,

marshy soils, and the rumen of cattle and sheep is a rep-

resentative species of this domain. Methane was fi rst ob-

served as a type of combustible air by the Italian physicist

Alexandro Volta in 1776 who collected gas from marsh

sediments and showed that it was fl ammable (the Volta

experiment). Later it was subsequently discovered by Bee-

hamp, Popoff, Tappeneiner, Hoppe-Seyler, Sohugen and

Omelianski that certain microbial species were responsible

for the production of methane. In earlier taxonomic treat-

ments methanogens were grouped among the better charac-

terized bacterial group on the basis of their morphologies.

Later they were clustered into a single family Methanobac-

teriacea. Till now a wide variety of methanogens have been

described, their taxonomy based on both phenotypic as well

as phylogenetic analysis (comparative 16 S rRNA sequenc-

ing), with several orders being recognized.

Classifi cation of methanogens

The biological classifi cation is a hierarchical system that

starts with a few categories at the highest level at further

S. K. Sirohi1 (�) · N. Pandey

1 · B. Singh

1 · A. K. Puniya

2

1Nutrition Biotechnology Lab, Dairy Cattle Nutrition Division,

National Dairy Research Institute,

Karnal - 132 001, India

2Senior Scientist, Dairy Microbiology Division,

NDRI, Karnal

E-mail: [email protected]

254 Indian J Microbiol (September 2010) 50:253–262

123

subdivides them at each lower level. The methanogenic

bacteria are divided into three orders which are further sub

divided into family, genus and species [2].

I Order Methanobacteriales Are very strict anaerobes

and are found in anaerobic habitats such as sediments of

natural waters, soil, anaerobic sewage digester, gastrointes-

tinal tract of animals (rumen). These consists of short lancet

shaped cocci to long fi lamentous rods, typically gram posi-

tive but some cells may be gram variable.

Pseudomurien is the main cell wall content. Substrates

utilized as energy source are hydrogen, formate or CO. Any

other organic material is not utilized.

Family-Methanobacteriacae consists of generas Metha-

nobacterium and Methanobrevibacter

The Genus Methanobacterium consists of curved,

crooked to straight non sporing rods, long and fi lamentous

about 0.5–1.0 μm in width. Non motile or motile due to

fi mbrae, very strict anaerobes, mesophiles to extreme ther-

mophiles.

Examples of species included in this genus are M. formi-

cicum, M. bryantii M. thermoautotrophicum

The Genus Methanobrevibacter consists of short rods

or lancet shaped cocci which often occurs in pairs or chain

(0.5–1.0 μm in width).Cells are poorly motile or non motile

with optimal growth range at 37–39°C.

Examples of species included in this genus are M. rumi-

nantium, M. smithi, and M. aboriphilus

II Order Methanococcales consists of gram negative ir-

regular cocci. Cell wall consists of a single layer of protein;

H2 and formate are used as substrate for growth and metha-

nogenesis. Widely found in sediments of natural waters.

Family Methanococcacae consists of genus Methano-

coccus.

Genus Methanococcus consists of regular to irregular

cocci which may be single or paired, cells are highly mo-

tile.

Examples of species included in this genus are M. voltae,

M. vannieli

III Order Methanomicrobiales consists of rods or coc-

cus which may be gram positive or gram negative, motile

or non-motile. They oxidize hydrogen or formate with

reduction of CO2 to CH

4 via fermentation of methanol, me-

thylamine (trimethylamine and ethyl dimethylamine), and

acetate. Does not utilize any carbohydrate, proteinaceous

material or organic compound. It is widely distributed in

nature. Found in sediments of natural waters, soil, an-

aerobic sewage digester and GI tract of animals (rumen).

A. Family-Methanomicrobiacae consists of gram negative

cocci or slightly curved or straight rods. Oxidize hydrogen

or formate as sole energy source for growth and methane

production.

i Genus Methanomicrobium consist of short, straight

or slightly curved rods with rounded ends. Cells are motile.

Optimum temperature for growth is 38–40°C. Only hydro-

gen serves as substrate for growth and methane production.

Examples of species included in this genus are M. mo-

bile

ii Genus Methanogenium consists of gram negative,

irregular coccoid cells that may or may not require acetate.

Species included in this genus are M. cariaci, M. maris-

nigri

iii Genus Methanospirillum consists of slender rods

that continuously form spiral fi lament, which are motile.

Example of species included in this genus is M. hunga-

tei

B. Family Methanosarcinacae consists of large, spheri-

cal to pleomorphic gram positive cells (1.5 to 2.5 μm in

diameter), often forming packets of various sizes. Planes of

division are not always perpendicular. Cells are non-motile,

mesophiles to thermophiles. Oxidise hydrogen with reduc-

tion of CO2 to CH

4 by metabolism of methanol, methyl-

amine (di, tri, ethyldimethyl) and acetate. Methane, carbon

dioxide and ammonia are formed as end products. Cell wall

consists of heteropolysaccharide.

Examples of Genus Methanosarcina is M. barkeri

Cell envelopes or cell wall of Methanogens

Methanogens exhibit great diversity in cell envelopes,

ranging from simple, nonrigid surface layers consisting of

protein or glycoprotein subunits to a rigid “pseudomurein”

sacculus, analogous to eubacterial murein [3–6]. Muramic

acid or D-amino acids have not been detected till date. Ac-

cording to their major cell wall constituent, cell envelopes

of Methanogens may be categorized into three characteris-

tic classes: (i) pseudomurein layer (ii) protein or glycopro-

tein layer; and (iii) heteropolysaccharides layer.

Order methanobacteriales

Members of the gram-positive methanobacterium consists

of sharply defi ned, smooth cell wall 15–20 nm in width and

are the only archaebacterial species that possess a pseudo-

murein-type cell wall analogous to eubacterial murein [7,

4]. Pseudomurein differs from eubacterial murein in that (i)

L-talosaminuronic acid is substituted for muramic acid (ii)

different sequences of amino acids (L confi guration of ala-

nine, glutamic acid, and lysine, mainly) are constituents of

peptides involved in the glycan polymer cross linking (iii)

the chemical bonds between the sugar moieties of alternat-

ing N-acetylglucosamine and N-acetyl-talosaminuronicacid

are probably 1(1-3) linkages instead of β (1-4) glycosidic

linkages which occur in eubacterial murein [8, 6].

123


Antibiotics such as Vancomycin and Penicillin, which

affect eubacterial cell wall biosynthesis by interfering with

reactions involving D-alanine, do not affect biosynthesis

of methanogen pseudomurein [9]. Eubacterial murein and

methanogen pseudomurein appear to be analogous based on

function, chemical composition, and primary structure.

In the genus Methanobrevibacter, ultra thin section of

whole cell of M. ruminantium reveal that the cell is cov-

ered by a triple layered cell wall of width 30–40 nm. The

inner layer is an electron dense layer; the middle layer is

electron transparent layer while the outer layer is rough and

irregular. The cell wall contents also differed in polypeptide

sequence, as the l-ala is replaced by l-threonine and NAG

is replaced by N-acetyl galactosamine. It also contains high

phosphate level in its cell wall content.

Order Methanococcales

Cell wall consists of a layer of protein or glycoprotein sub-

units (S-layer) with traces of glucosamine external to the

cell membrane. In many species it is lysed easily [10] by

detergents or solutions of low osmolality. However, some

species posses detergent resistant and protease resistant

S- layer [11]. The carbohydrate components of the glyco-

protein in S-layers vary greatly. No muramic acids or DAP

have been yielded from cells. Traces of glucosamine have

been found in M. vanielli and M.voltae. One member of the

Methanobacteriales, the extremely thermophilic Methano-

thermus fervidus, has a pseudomurein cell envelope covered

by a layer of protein subunits. Perhaps the S-layer provides

greater thermostability for Methanothermus fervidus and is

an adaptive trait in response to environmental factors.

Electron micrographs of the outer surface of Methano-

coccales spp showed a regular array of protein subunits. Ul-

tra thin sections of whole cell revealed a single layer of cell

wall material 18 nm thick (S-layer). On treating cells with

2% SDS at 100°C for 30 min. or disintegration of cells with

glass beads followed by incubation with trypsin resulted in

complete solublization.

S-layers are not unique to archaebacteria; in fact, pro-

tein-containing S-layers are found in diverse species of eu-

bacteria, and little difference in the chemical compositions

of S-layers of eubacteria and archaebacteria have been de-

tected. In general, S-layers from both groups are composed

of proteins which are rich in acidic amino acids and have a

low percentage of sulfur-containing amino acids.

Order Methanomicrobiales

This order consists of Heteropolysaccharide and com-

plex cell envelopes (Third distinguishing cell wall type

found in archaebacteria is restricted to Halococcus and

Methanosarcina spp.). A thick, amorphous cell wall structure

consisting of acid heteropolysaccharide containing galactos-

amine, neutral sugars and uronic acids is found in methano-

gens belonging to the genus Methanosarcina, which usually

grow in spherical packets. The structural wall in Methano-

sarcina is a polymer of D-glucuronic acid and N-acetylga-

lactosamine, which is similar to animal chondroitin [4]. The

constituents of this polymer in Methanosarcina sp. are not

sulfated as in the case of Halococcus sp. Zeikus et al reported

that the outer layer appears to be laminated. Zhilina reported

a triple layered appearance of cell wall in gas vacuolated

strain. They lack the constituents of peptidoglycan [3].

Ultra thin sections of genus Methanogenium showed a

cell wall of 10nm which consisted completely of protein

confi rmed by freeze drying the cells and treating with SOD

or treating the disintegrated cells with trypsin resulting

in complete solublization. No muramic acids or amino

sugars were detected [3]. Methanospirillum hungatei and

Methanothrix soehngenii are characterized by complex cell

envelopes containing a thin, fi brillar outer sheath surround-

ing an electron-dense inner wall [12, 13] which covers the

cells separated by spacer elements. Isolated sheath material

consists of protein (18 amino acids and is resistant to SOD

or trypsin treatment) and possibly glycoprotein, as indicated

by the presence of amino acids and neutral sugars as sheath

hydrolysis products [3, 4]. The inner wall of Methanothrix

sp. is involved in septum formation during cell division as

indicated by electron microscopy; this phenomenon has not

been observed in Methanospirillum spp. No muramic acids

or amino sugars have been detected [3]. Little else is known

about this unusual cell wall structure.

In case of methanogens a positive gram reaction is seen

if there is the presence of a thick rigid sacculus whereas

gram negative reaction reveals its absence [3].

Methanogens found in rumen

Methanogenesis in ruminants has important environmental

consequences. Methanogens such as Methanomicrobium

mobile, Methanobacterium formicicum, M. bryantii, Meth-

anobrevibacter ruminantium, M. smithi, Methanosarcina

barkeri, and M. mazai have been isolated from rumen by

cultural methods. However, molecular methods reveal a

considerable genetic diversity of methanogens in the ru-

men, even within the same ruminant species. Some of the

methanogens are non-culturable.

Characteristics of M. formicicum

Cell shape Slender, cylindrical with blunt rounded ends.

Some cells are unevenly crooked. Some chains and fi la-

ments are seen.


123

Cell size Depending upon the strain and length the cell

width may vary from 0.4–0.8 μm.

Motility Non motile.

Gram nature Gram variable.

Major lipids:

a. neutral (isoprenoid hydrocarbons) Not determined.

b. polar (isopranyl glycerol ethers) C20

+ C40

ethers.

Substrate for growth and methanogenesis H2 and formate.

Acetate, carbohydrate, aminoacid, ethanol, methanol, pro-

pionate, butyrate and lactate are not fermented. CO may

be fermented in some strains. Some strains do not utilize

formate.

Optimum temperature for growth are mesophiles, with

optimum growth at 38 and 45°C. No growth at 55°C.

Surface colony characters Colonies are white to gray,

fl at and fi lamentous; deep colonies appear as profusely

fi lamented sphaeroid. Incubation period of 3–5 days and

temperature of 37°C is required. In about 14 days colonies

attain a diameter of 2–5 mm.

Growth in liquid broth Depending upon the strain, either

turbidity or granular clumps are seen which do not break

even with vigorous agitation.

Host Bovine, ovine.

Characteristics of M. bryanytii

Cell shape Slender, cylindrical with blunt rounded ends,

often forming chains or fi laments with unevenly crooked

cells.

Cell size Chains may be up to 10–15 μm in length. Cell

width varies from 0.5–1.0 μm.

Motility Non motile and posses fi mbrae.

Gram nature Gram positive to Gram variable.

Major lipids:

a. neutral (isoprenoid hydrocarbons) C30

H50

, C30

H52.


+ C40

ethers.

Substrate for growth and methanogenesis H2 is only uti-

lized. Formate is not used. Ammonium ion is essential as

source of nitrogen. Acetate, cystiene and B-vitamins are

stimulatory for growth.

Optimum temperature for growth 37–39°C.

Optimum pH for growth 6.9–7.2.

Surface colony characters Gray to light gray colonies

which are fl at with diffuse to fi lamentous edges. They can

reach a diameter of 1–5 mm.

Host Bovine.

Characteristics of M. ruminantium

Cell shape Very short lancet shaped to oval rods or coc-

cus, may occur in pairs but usually in chains which may be

up to 20 or more cells resembling Streptococci.

Cell size Cells are 0.5–1.0 μm in width and 1.0–1.5 μm

in length.


Gram nature Gram positive, even in relatively old cul-

tures.

Major lipids:


H50

, C30

H52

,

C30

H54

,C30

H56.


+ C40

ethers.


Carbohydrate, amino acid, methanol, ethanol, isobutyrate,

propionate, valerate, caproate, succinate and pyruvate are

not utilized. Acetate, ammonia and sulfi de may be essential

as important source of cell carbon, nitrogen and sulfur. One

or more vitamins are required. It also requires 2-methyl n-

butyrate and CoM.

Optimum temperature for growth 37–43°C. Little or no

growth at 47°C. Rumen strains do not grow at 33°C.

Optimum pH for growth 6.3–6.8.

Surface colony characters Colonies are off white to

yellow in color, translucent, convex and circular with

entire margins. They become visible after 3 d of incubation

at 37°C and may reach a diameter of 3–4 mm depending

upon number of colonies. Colonies in deep agar are len-

ticular.

Growth in liquid broth Growth is turbid or fl oc. Vigorous

shaking results in breaking of fl ocs.

Host Bovine.

Characteristics of M. smithi

Cell shape Morphologically similar to M.ruminantium,

with the exception of a single polar fl agellum. Short, lancet

shaped oval cocci; may occur in pairs or chain.

Cell size Cells are 0.5–1.0 μm in width and 1.0–1.5 μm

in length.


Gram nature Strongly Gram positive.

Major lipids:


H50

, C30

H52.


+ C40

ethers.

Substrate for growth and methanogenesis Either H2 or

formate. Can be cultured in simple chemical defi ned media,

which differentiates it from M.ruminantium. Ammonia and

acetate are required as major source of cell nitrogen and

carbon.

Optimum temperature for growth 37–39°C .

Optimum PH for growth 6.9–7.4.

Surface colony characters Yellow to white colonies that

are translucent, convex, and circular with entire margins.

Host Bovine.

123


Characteristics of M. mobile

Cell shape Straight to slightly curved rods with rounded

ends. No chain is seen. May occur as single or in pairs.

Cell size Are 0.7 μm wide and 1.5–2.0 μm long.

Motility Motile with monotrichous fl agella.

Gram nature Gram negative.

Major lipids:

a. neutral (isoprenoid hydrocarbons) Not determined.

b. polar (isopranyl glycerol ethers) Not determined.


Acetate, propionate, butyrate, isobutyrate, valerate, isova-

lerate, caproate, succinate, glucose, pyruvate, methanol,

ethanol, propanol, isopropanol, and butanolare not utilized

as electron donors. It can grow well on media devoid of ru-

men fl uid or extracts of mixed ruminal bacteria.

Optimum temperature for growth 38–40°C.

Optimum PH for growth 5.9–7.7.

Surface colony characters Colorless to pale yellow colo-

nies which are small, translucent, entire and convex. They

require an incubation period of 4 d at 39°C and reach a di-

ameter of 0.7–1.0 mm in 15 d. Deep colonies are lenticular

and 0.5–0.7 mm in diameter in 15 d.

Host Bovine.

Characteristics of M. barkerii

Cell shape Are spheres mostly occurring in packets of

eight or less but sometimes in large mass.

Cell size The diameter of the sphere is 1.5–2.5 μm.


Gram nature Gram positive.

Major lipids:


H46

, C25

H48

,

C25

H50

, C25

H52.


ethers.

Substrate for growth and methanogenesis H2 is used.

Methanol, methylamine, acetate and CO are fermented with

the formation of CH4 and CO

2. Growth and methanogenesis

are more rapid in methanol than in acetate media. Carbohy-

drate, amino acid, formate, ethanol, propionate, butyrate are

not fermented.

Optimum temperature for growth Are mesophiles to ther-

mophiles.

Optimum PH for growth 7.0.

Surface colony characters Deep colonies are whitish and

0.5–1.0 mm in diameter in methanol agar with inorganic

salts.

Growth in liquid broth Growth may occur as zoogloeal

masses on as fl occulent sediments with active gas formation.

Host Carpine, bovine

Both CoM requiring and non requiring strains of Metha-

nobrevibacter have been isolated from bovine rumen [14,

15]. Lovely et al. [14] isolated two strains of Methanobrevi-

bacter from high dilutions of bovine rumen fl uid. The Co M

Synthesizing strain had simple nutritional requirements and

higher growth rate as compared to non synthesizing strain;

also it did not reacted with the antiserum against type strain

of M. rumination. Similarly four CoM requiring and two

CoM non requiring strains of M. ruminantium have been

isolated by Miller et al. [15]. However none of the strain re-

acted with antiserum against the type strain of M. ruminan-

tium. These observations confi rm that both CoM requiring

and non requiring strains of M. ruminantium are present in

high concentration in bovine rumen content.

Culture of methanogens

Methanogens require very low redox potential and are

perhaps the strictest anaerobic bacteria known. They can

be cultured only by procedures that ensure culture in the

absence of oxygen. Many workers have developed proce-

dures for cultivating Methanogens. The Hungate culture

technique, with modifi cations, has proven to be an excel-

lent method for isolating fastidious anaerobes. The methods

described by Bryant [16] for culturing larger quantities of

cells have proven acceptable for most Methanogenic spe-

cies. Bryant et al. [16, 17] cultured methanogens using glass

test tubes (anaerobic culture tubes) that are tightly sealed

with neoprene, butyl, or synthetic, but not gum, rubber stop-

pers. A gas mixture of 80% H2 and 20% CO

2 is used which

is made free of oxygen by passing it through heated copper

fi lings. Some investigators also prefer a 50:50 mixture of

H2 and CO

2 [16] because this gas mixture is more dense and

not as easily displaced by air when culture containers are

opened. The tubes are gassed while incubation or substrate

addition. Macy et al. [18] described the syringe method

for substrate or media addition or inoculation in “Hungate

type” tubes that are screw-capped and sealed with fl anged

rubber stoppers.

Serum bottles or various glass containers fi tted with

serum bottle necks have been described for cultivation of

methanogens by Miller and Wolin [19]. The bottles are

sealed with metal seal after closing with butyl rubber stop-

pers. This is advantageous over non sealed stoppers because

non sealed stoppers are often blown out of culture tubes as

the result of active fermentation of methanol. Here all inoc-

ulations and transfers are done with a hypodermic syringe

and needle. This technique is also better due to less fragility

and easy handling of serum bottles.

Use of Freter type anaerobic glove box equipped with

an inner ultralow oxygen chamber has been described by


123

Edwards and McBride [20] for isolation and growth of

methanogens. The inner chamber maintains the redox po-

tential necessary for growth of methanogens and is used for

incubation of plates in pressure cooker containers which are

specially modifi ed for high atmospheric pressure of H2 and

CO2 as the inner chamber, is periodically fl ushed with H

2

and CO2 (80:20). Cultures are plated in the outer anaerobic

glove box and immediately placed in the inner chamber.

This method is considerably more expensive than Hungate

procedures; however, it offers unique advantages. For ex-

ample, it requires less skill and manual dexterity, and it al-

lows for routine genetic procedures such as replica plating.

Balch and Wolfe [21] have described a new method for

cultivating H2 oxidizing methanogens by applying high gas

pressure (2 to 4 atmospheres of H2 and CO

2). This avoids

the need of gassing the culture repeatedly. Specialized gas-

sing manifold, glass culture tubes (for liquid cultures) and

anaerobic incubators (for agar plate cultures) have also been

used by these workers for isolating the organism.

Herman Knoll and Wolfe [22] described the isolation of

methanogenic bacteria using agar bottle plate. The bottle

solved the problem of water exudates from agar medium

and provided convenience of streaking, adding or sampling

a defi ned gas atmosphere.

Identifi cation and Quantifi cation of Methanogenic

Bacteria

F420

present in methanogens can be exploited to identify the

bacteria. Methanogenic colonies fl uoresce when exposed

to long wavelength UV radiations due to the presence of

cofactor F420

[20]. During the active growth of methanogens

the F420

exists in particular oxidized state [20]. This oxidized

form is excited by the long wavelength UV radiations re-

sulting in fl uorescence. Results of fl uorescent microscopy

can be enhanced by a proper selection of excitation and bar-

rier fi lters. Also, methanogenic bacteria can be identifi ed by

their bright fl uorescence under UV microscopy [23].

Gas Chromatographic Analysis (GC) is also used for

identifi cation and quantifi cation of methanogens. Isolated

cultures of methanogens actively produce methane by oxi-

dation of H2 and reduction of CO

2.Tubes containing broth

are inoculated, incubated and tubes are then observed for

methane as head gas by gas chromatography. This method

is also used for counting methanogens by MPN (Most Prob-

able Number). For MPN analysis tubes of three consecutive

dilutions are inoculated in triplicate. After incubation the

tubes containing more than 100 ppm of methane are count-

ed as positive. A more rapid, sensitive, and convenient GC

procedure for analysis of 4C-labeled and unlabeled metabol-

ic gases has also been described [24]. In this method gases

are detected by thermal conductivity detector and the effl u-

ent is directly channeled into a gas proportional counter for

radioactivity measurement. Thermal conductivity detection

is often more useful than fl ame ionization detection because

H2, CH

4, and CO

2 can be accurately quantifi ed on the same

column, where as Flame Ionization Detector is only limited

to CH - containing compounds.

Real-time polymerase chain reaction (PCR) using a

broad-range (universal) probe and primers set is also used

for the quantifi cation of methanogens [25].

Single Strand Conformation Polymorphism (SSCP)

based genetic probes of small subunit rRNA genes have

been described [26]. DNA is extracted from rumen fl uid

collected from cow and amplifi ed with Ex Taq DNA poly-

merase (TAKARA). Primers used for PCR are M301F

and M915R; Ar1000F and Ar1500R. The PCR product is

purifi ed and digested by exonuclease to yield ssDNA. This

ssDNA is reamplifi ed by PCR and is then sequenced. Ap-

plying SSCP 22 clones were sequenced.

FISH (Fluorescent in situ Hybridization technique)

This is one of the modern methods to study the complex

microbial communities. Here rRNA targeted fl uorescent

oligonucleotide probes are used. Whole cell FISH is de-

tected by confocal scanning laser microscopy (CSLM)

[27]. A new method of quantifi cation for Methanogens by

fl uorescence in situ hybridization (FISH) based on the mea-

surement of specifi c binding (hybridization) of 16S rRNA-

targeted oligonucleotide probe Arc915, has been described

by Stabnikova et al. [28]. Specifi c binding of probe per 1 ml

of microbial sludge suspension from anaerobic digester lin-

early correlated with concentration of autofl uorescent cells

of Methanogens. However this method is not applicable for

diluted suspensions of Methanogens.

The detection and quantifi cation of Methanogens by

the above mentioned methods have certain limitations like

there are methanogenic bacteria that have no F420

or only

low levels (The genus Methanosaeta) [29] or, conversely,

there are non methanogenic bacteria that exhibit similar

UV fl uorescence. Therefore further proof is required for

proper identifi cation. Also this method is not applicable

for aggregate forming cells (The genus Methanosarcina)

[30]. The plate counting method and the MPN method is

time consuming [31, 32] as Archaea are slow grower. Also

it requires special laboratory equipment and can evaluate

only viable cells. The Real time (quantitative) PCR method

is not suitable for dense suspension of methanogens. The

disadvantage of FISH described by Amman et al is extrac-

tion of RNA and use of radioactive labels.

Methanogenesis

Methanogenesis is the production of methane by methano-

genic bacteria by utilizing simple substrate at low reduction

123


potential, to produce cellular energy. Although some Eu-

bacteria have also been reported to produce methane [33],

only methanogens have been reported to couple methane

generation to energy production. Less than 1 ATP is derived

by cells from each molecule of methane produced. Some

unique enzymes are present in methanogens which carry

out the process of methanogenesis. Ralph Wolfe in early

1970s fi rst started studying the methanogenic reduction

of CO2 [34]. In the next 20 years six new coenzymes were

discovered. By the early 1990s the pathway of methanogen-

esis was elucidated. The process of methanogenesis (Fig. 1)

requires seven coenzymes and eight enzymes.

The various coenzymes involved in methanogenesis are

Coenzyme 420 – the (N-(N-L-lactyl-y-glutamyl) L-glutam-

Fig. 1 Methanogenesis by the reduction of CO2. (Elucidation of methanogenic coenzyme biosyntheses: from spectroscopy to genomics

David E. Graham and Robert H. White)2.


123

ic acid phosphodiester of 7, 8-didemethyl) 8-hydroxy-5-

deazaribofl avin 5 phosphate was fi rst isolated from Metha-

nobacterium strain M.o.H. It has an absorption maxima at

420nm and hence its name. It gives blue green fl uorescence

in oxidized state. The fl uorescence is lost upon reduction. It

is present in methanogens at levels ranging from 1.2 mg/kg

of dry cell wt. in M.ruminantium to 65 mg/kg of dry cell wt.

in M. thermoautotrophicum.

Earlier this coenzyme was believed to be unique to hy-

drogen metabolizing euryarchaea. Later it was also identi-

fi ed in Halobacteria and some Gram positive bacteria. All

the F420

found in these organisms differ in their glutamate

side chains though they are functionally interchangeable

and have comparable spectroscopic properties. Some

cyanobacteria, mosses, green algae, midge produce DNA

photolyase containing 8-hydroxy 5-deazaribofl avin cofac-

tor. F420

with additional glutamyl residue linked to distal

glutamate is found in M. barkerii [36].

F420

is a low potential electron carrier [38]. The reduc-

tion potential of F420

/F420

-H2 is -340to -350mV, between the

redox potential of NAD (P)/NAD (P) H and 2H+/H2. It is

similar to nicotinamide cofactors as it functions in two elec-

tron transfer reactions. It also shows some similarities with

ribofl avin as both ribofl avin and F420

are synthesized from a

shared precursor, also the 8 hydroxy 5 deazaribofl avin moi-

ety of F420

is structurally similar to ribofl avin.

The activities of formate dehydrogenase [29, 41], CO

dehydrogenase [37], hydrogenase [38–40] NADP

+ reduc-

tase [39, 41, 42], pyruvate synthetase and α-ketoglutarate

synthetase [43,44] are coupled to oxidation /reduction of

F420

.

F390

-In oxygen stressed methanobacterium cells a new

chromophore derived from F420

was discovered [45]. Struc-

turally they are F420

adducts. They have adenosine-5 phos-

phate /guanosine-5 phosphate linked to 8 hydroxy groups.

They act as alarmone [45] in response to oxygen stress.

They also activate the enzyme NADP+ reductase and play

an important role in metabolism.

Coenzyme M – The CoM (2-mercaptoethanesulfonic

acid) is the smallest known organic cofactor. It was fi rst

characterized in 1971 in Methanobacterium strain M.o.H.

as one of the several enzymes involved in methanogenesis

[46]. Taylor and Wolfe described the structure of oxidized

(S-Co M)2 disulfi de. Till 1999 it was considered unique to

methanogens until it was discovered in Xanthobacter as a

cofactor in alkane oxidation pathway. It acts as terminal

methyl carrier in methanogenesis.

Coenzyme B – It is a colorless cofactor. It was earlier

called component B as it was identifi ed as one of the three

chromatographically separated fractions required to recon-

stitute MCR (Methyl Coenzyme reductase). Its structure

was determined as 7-mercaptohepta-moylthreonine phos-

phate. It acts with Coenzyme M in the fi nal step of metha-

nogenesis. It contains a thiol group and an L-threonine

phosphate group which is specifi cally recognized by MCR.

The thiol group displaces methane from M methyl Co M

and L- threonine phosphate group binds to basic amino acid

in MCR.

Methanofuran (MFR or carbon diooxide reduction

factor) – It was fi rst obtained from the cell extracts of

Methanobacterium thermoautotrophicum. Earlier it was

named as CO2 reduction factor [47]. Later its structure was

determined and was renamed as Methanofuran [48]. It is

the only cofactor known to contain furan moiety. It is found

in all methanogens at level ranging from 0.5–2.5 mg/kg of

cell dry wt–[49]. Five different Methanofuran cofactors are

produced by Archaea. The central core structure consist

of 4-[N-(7-L-glutamyl-7-L-glutamyl)-P-(B-amino-ethyl)

phenoxy methyl]-2-(amino methyl) furan, to which ad-

ditional structures are attached by an amide bond to the ά-

amino of terminal glutamyl residue. The MFR isolated from

Methanobacterium thermoautotrophicum consisted of core

structure attached to 1, 3, 4, 6-hexanetdra carboxylic acid

(HTCA). MFR found in Methanosarcani barkeri consist of

2 ү-linked glutamic acid [19] while to hydroxyl HTCA is

found in MFR found in M. smithi.

MFR react with CO2 in the fi rst of

methanogenesis and

forms an N- carboxymethanofuran. This cabamate is re-

duced to formyl methanofuarn by enzyme formyl methano-

furan dehydrogenase.

Methanopterin- It was originally identifi ed in Metha-

nobacterium thermoautotrophicum and Methanosar-

cina barkeri. The cofactor in Methanosarcina barkeri was

known as sarcinopterin. The structures of both the cofac-

tor are similar except their dicarboxylic acid side chains

sarcinopterin contain a glutamyl residue esterifi ed to the

hydroxyl glutarate moiety.

Structurally Methanopterin is related to folic acid [8,

50] as it consists of the same pteroic acid core found in

folates. Pteroic acid core is attached to L-glutamates in

folates, whereas methanopterin consists of pteroic acid core

whose carboxylic acid is replaced by ribitol containing side

chain also, the reduced form of both the cofactors, H4MPT

and H4F are biologically active substances. However, both

are well differentiated by enzymes that are specifi c for

them.

In the process of methanogenesis MPT acts as inter-

mediate C1 carrier in the reduction of formyl group to

methyl group. H4MPT is also required by M. thermoau-

totrophicum to synthesize acetate, whereas the methylene

tetrahydromethanopterin is required for serine synthesis

[51].

123


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Rumen methanogens: a review

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